Inhibition Of Breast Cancer MDA-MB-231 Cells By Nitidine Chloride And Its Preliminary Study Of Anti-breast Cancer Through COL8A1

Objective:(1)In vitro cell experiments were conducted to investigate the inhibitory effect of Nitidine chloride(NC)on breast cancer(BC),and provide experimental basis for its mechanism research.(2)Through computational biological analysis methods,the mechanism of action of NC anti-BC is explored at the molecular level,providing ideas for finding potential biological targets of NC.(3)The functional significance of the NC potential target gene COL8A1 in BC is unclear.By detecting the expression level of COL8A1 in BC,and analyzing its relationship with clinicopathological parameters and prognosis,it provides clinical data support for NC molecular mechanism research.(4)By constructing BC cell COL8A1 overexpression transfected cell line and cell function experiments,it was analyzed whether NC inhibits BC progression by regulating COL8A1.Materials and Methods: 1.Proliferation and migration of BC cells after NC treatment by CCK8 cell proliferation assay,scratch assay and plate cloning assay.2.(1)By collecting Gene Expression Omnibus(GEO)NC treatment of BC beforeand after chip data,screening Differentially Expressed Genes(DEGs)before and after NC treatment of BC cells,Gene Ontology(GO),the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis,Disease Ontology(DO)analysis,protein-protein interaction(PPI)analysis,and explore the potential molecular mechanism of NC against BC.(2)In addition,the Connectivity Map(CMAP)database was used to search for small molecule drugs similar to the expression profile of NC chip DEGs,and the pharmacological mechanism of NC anti-BC was analyzed.(3)In addition,23 sets of GEO Affymetrix Genechips and DEGs of BC in the Gene Expression Profiling Interactive Analysis(GEPIA)database were extracted,and then the intersection of DEGs of NC processing BC chips was taken to obtain potential target genes of NC in BC.(4)Real-time Quantitative polymerase chain reaction(RT-qPCR)was used to detect the mRNA expression of candidate genes in NC-treated BC cells,and the accuracy of the chip results was verified.3.(1)The expression of COL8A1 in 23 sets of breast cancer GEO Affymetrix Genechips and the Cancer Genome Atlas(TCGA)database was extracted.The mRNA expression of COL8A1 in BC and its ability to distinguish between BC and normal breast were analyzed.The mRNA expression of COL8A1 was discussed.Significance of BC patients with prognosis and prognosis.(2)Immunohistochemical method was used to detect the protein expression level of COL8A1 in paraffin-embedded specimens of 115 cases of BC and 65 normal breasts.The relationship between protein expression of COL8A1 and prognosis and clinicopathological features of BC patients was explored.4.(1)Lentiviral construction overexpresses COL8A1 stably transfected strain,and cell function experiments verify whether overexpression of COL8A1 affects NC anti-BCeffect.Result: 1.(1)Different concentrations of NC(0.5,1,2,4,8,16,32 μM / L)significantly inhibited the proliferation of MDA-MB-231 cells,and showed a significant concentration-time-dependent inhibition of cell proliferation.(P<0.05).The IC50 value of NC-treated MDA-MB-231 cells for 48 h was(2.05±0.78)μM/L.(2)The cell scratch test showed that the migration rate of cancer cells in the treatment group(1,2,4 μM/L)was significantly lower than that in the control group(P<0.05).(3)The cells were treated with NC(0,1,4μM/L)for 48 h,and the number of cell clone formation after 15 days of culture was(50.75±18.57),(10.50±2.65),(0±0),respectively.The number of clones decreased significantly with increasing NC concentration(P<0.01).2.(1)There were 1444 DEGs in NC-treated breast cancer GEO chips,including 654up-regulated genes and 790 down-regulated genes.Through the analysis of GO and KEGG pathways,these DEGs are found to be involved in biological processes and signaling pathways involved in cancer progression,including "extracellular matrix","extracellular matrix organization","extracellular matrix structural constituent","protein tyrosine kinase activity","PI3K-Akt signaling pathway","ECM-receptor interaction","Focal adhesion",etc.DO enrichment analysis showed that DEGs gene function is associated with diseases such as "female reproductive system diseases" and "reproductive system diseases".PPI analysis of up-and-down DEGs screens for 6 gene modules with high interaction.(2)NC treatment of breast cancer GEO chip DEGs was introduced into the CMAP database,and three anticancer drugs similar to NC anti-BC expression profiles were screened: irinotecan,kaempferol,etoposide.(3)3537 DEGs of BC were calculated in GEO chip,3557 DEGs were selected from TCGA and GTExsequencing data,and the DEGs of the above-mentioned NC intervention were separated from the DEGs of BC in GEO and TCGA,and 90 overlapping genes were obtained,72 of which were obtained.The gene was up-regulated after NC treatment,and 18 genes were down-regulated after NC treatment.After screening,three genes(SPC24,RELN,COL8A1)that may be NC targets in BC were obtained for verification.(4)RT-qPCR analysis showed that the relative expression of COL8A1 mRNA was 0.63 and 0.04 times of that of the control group after treatment with NC treatment(1,4μM/L)for 48 h,and the results were consistent with the NC processing of the GEO chip,and the indicator was selected as the target gene for further research.3.(1)A meta-analysis of the mRNA expression levels of the COL8A1 gene in the GEO and TCGA databases showed that the mRNA expression level of COL8A1 in BC tissues was significantly higher than that in normal tissues(SMD=0.79,95% CI: 0.58-1.03,P=0.047).The area under curve(AUC)of SROC plotted for COL8A1 was 0.83(95% CI: 0.79-0.86).Further correlation analysis of clinicopathological factors revealed that COL8A1 mRNA levels in TCGA data showed significant differences in race,ER,PR,and molecular subtypes(P<0.001).Kaplan-Meier analysis showed no significant correlation between COL8A1 mRNA changes and survival time in patients with BC(P=0.267).(2)Immunohistochemistry showed that COL8A1 protein level was highly expressed in BC compared with normal breast tissues(P<0.05).The expression of COL8A1 protein was significantly correlated with ER level in BC patients(P<0.05).4.(1)The expression of COL8A1 mRNA in lv-COL8A1 transfected cells was significantly higher than that in control group and lv-Control group(P<0.05),indicating that stably constructed cell lines were successfully constructed.(2)Overexpression ofCOL8A1 can promote the proliferation of MDA-MB-231 cells and partially resist the inhibition of proliferation and cloning of BC cells by NC.(3)Overexpression of COL8A1 had no statistically significant effect on NC inhibition of BC cell migration.Conclusion: 1.This study predicted the multiple signaling pathways and 90 potential target genes in NC treatment of BC cells by computational biology and CAMP method,which provided an important theoretical basis for the study of the molecular mechanism of NC anti-BC.2.COL8A1 was highly expressed in BC tissues,and COL8A1 overexpression promoted MDA-MB-231 cell proliferation;There is no significant correlation between the expression of COL8A1 and the prognosis of BC.3.NC inhibited the proliferation and migration of human BC cell MDA-MB-231 in vitro and significantly decreased its COL8A1 mRNA expression.NC may inhibit the growth and proliferation of BC cell MDA-MB-231 by down-regulating COL8A1 mRNA expression,but it has no effect on cancer cell migration.